7-Fluorosialyl Glycosides Are Hydrolysis Resistant but Readily Assembled by Sialyltransferases Providing Easy Access to More Metabolically Stable Glycoproteins.

The maintenance of therapeutic glycoproteins within the circulatory system is associated, in large part, with the integrity of sialic acids as terminal sugars on the glycans. Glycoprotein desialylation, either by spontaneous cleavage or through host sialidases, leads to protein clearance, mainly through the liver. Thus, the installation of minimally modified sialic acids that are hydrolysis-resistant yet biologically equivalent should lead to increased circulatory half-lives and improved pharmacokinetic profiles. Here we describe the chemoenzymatic synthesis of CMP-sialic acid sugar donors bearing fluorine atoms at the 7-position, starting from the corresponding 4-deoxy-4-fluoro-N-acetylhexosamine precursors. For the derivative with natural stereochemistry we observe efficient glycosyl transfer by sialyltransferases, along with improved stability of the resultant 7-fluorosialosides toward spontaneous hydrolysis (3- to 5-fold) and toward cleavage by GH33 sialidases (40- to 250-fold). Taking advantage of the rapid transfer of 7-fluorosialic acid by sialyltransferases, we engineered the O-glycan of Interferon α-2b and the N-glycans of the therapeutic glycoprotein α1-antitrypsin. Studies of the uptake of the glyco-engineered α1-antitrypsin by HepG2 liver cells demonstrated the bioequivalence of 7-fluorosialic acid to sialic acid in suppressing interaction with liver cell lectins. In vivo pharmacokinetic studies reveal enhanced half-life of the protein decorated with 7-fluorosialic acid relative to unmodified sialic acid in the murine circulatory system. 7-Fluorosialylation therefore offers considerable promise as a means of prolonging circulatory half-lives of glycoproteins and may pave the way toward biobetters for therapeutic use.

Design, Synthesis, and Evaluation of (2-Aminocyclopropyl)phenyl Derivatives as Novel Positron Emission Tomography Imaging Agents for Lysine-Specific Demethylase 1 in the Brain.

Dysregulation of histone H3 lysine 4 (H3K4) methylation is implicated in the pathogenesis of neurodevelopmental disorders. Lysine-specific demethylase 1 (LSD1) determines the methylation status of H3K4 through flavin adenine dinucleotide (FAD)-mediated histone demethylation. Therefore, LSD1 inhibition in the brain can be a novel therapeutic option for treating these disorders. Positron emission tomography (PET) imaging of LSD1 allows for investigating LSD1 expression levels under normal and disease conditions and validating target engagement of therapeutic LSD1 inhibitors. This study designed and synthesized (2-aminocyclopropyl)phenyl derivatives with irreversible binding to LSD1 as PET imaging agents for LSD1 in the brain. We optimized lipophilicity of the lead compound to minimize the risk of nonspecific binding and identified 1e with high selectivity over monoamine oxidase A and B, which are a family of FAD-dependent enzymes homologous to LSD1. PET imaging in a monkey showed a high uptake of [18F]1e to regions enriched with LSD1, indicating its specific binding to LSD1.

Synthesis and in†vivo Evaluation of Fluorine-18 and Iodine-123 Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine Derivatives as PET and SPECT Radiotracers for Mapping A2A Receptors.

Imaging agents that target adenosine type 2A (A2A ) receptors play an important role in evaluating new pharmaceuticals targeting these receptors, such as those currently being developed for the treatment of movement disorders like Parkinson's disease. They are also useful for monitoring progression and treatment efficacy by providing a noninvasive tool to map changes in A2A receptor density and function in neurodegenerative diseases. We previously described the successful evaluation of two A2A -specific radiotracers in both nonhuman primates and in subsequent human clinical trials: [(123) I]MNI-420 and [(18) F]MNI-444. Herein we describe the development of both of these radiotracers by selection from a series of A2A ligands, based on the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine core of preladenant. Each of this series of 16 ligands was found to bind to recombinant human A2A receptor in the low nanomolar range, and of these 16, six were radiolabeled with either fluorine-18 or iodine-123 and evaluated in nonhuman primates. These initial in vivo results resulted in the identification of 7-(2-(4-(4-(2-[(18) F]fluoroethoxy)phenyl)piperazin-1-yl)ethyl)-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine ([(18) F]MNI-444) and 7-(2-(4-(2-fluoro-4-[(123) I]iodophenyl)piperazin-1-yl)ethyl)-2-(furan-2-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-amine ([(123) I]MNI-420) as PET and SPECT radiopharmaceuticals for mapping A2A receptors in brain.

Identification and in vivo evaluation of a fluorine-18 rolipram analogue, [(18) F]MNI-617, as a radioligand for PDE4 imaging in mammalian brain.

Phosphodiesterase (PDE) 4 is the most prevalent PDE in the central nervous system (CNS) and catalyzes hydrolysis of intracellular cAMP, a secondary messenger. By therapeutic inhibition of PDE4, intracellular cAMP levels can be stabilized, and the symptoms of psychiatric and neurodegenerative disorders including depression, memory loss and Parkinson's disease can be ameliorated. Radiotracers targeting PDE4 can be used to study PDE4 density and function, and evaluate new PDE4 therapeutics, in vivo in a non-invasive way, as has been shown using the carbon-11 labeled PDE4 inhibitor R-(-)-rolipram. Herein we describe a small series of rolipram analogs that contain fluoro- or iodo-substituents that could be used as fluorine-18 PET or iodine-123 SPECT PDE4 radiotracers. This series was evaluated with an in vitro binding assay and a 4-(fluoromethyl) derivative of rolipram, MNI-617, was identified, with a five-fold increase in affinity for PDE4 (Kd = 0.26 nM) over R-(-)-rolipram (Kd = 1.6 nM). A deutero-analogue d2 -[(18) F]MNI-617 was radiolabeled and produced in 23% yield with high (>5 Ci/µmol) specific activity and evaluated in non-human primate, where it rapidly entered the brain, with SUVs between 4 and 5, and with a distribution pattern consistent with that of PDE4.

Change in PDE10 across early Huntington disease assessed by [18F]MNI-659 and PET imaging.

OBJECTIVE: To evaluate whether striatal [(18)F]MNI-659 PET imaging of phosphodiesterase 10A (PDE10) serves as a sensitive and reliable biomarker of striatal neurodegeneration in a longitudinal cohort of participants with early Huntington disease (HD). METHODS: A cohort of participants with HD, including both participants premanifest or manifest with motor signs, underwent clinical assessments, genetic determination, and 2 [(18)F]MNI-659 PET imaging sessions approximately 1 year apart. Eleven healthy control (HC) participants underwent clinical assessments and [(18)F]MNI-659 PET imaging once. Striatal binding potentials (BPnd) were estimated for brain regions of interest, specifically within the basal ganglia, and compared between baseline and follow-up imaging. Clinical measures of HD severity were assessed at each visit. RESULTS: Eight participants with HD (6 manifest; 2 premanifest) participated. Of those with manifest HD, all had relatively early stage disease (stage 1, n = 2; stage 2, n = 4) and a Unified Huntington's Disease Rating Scale total motor score <45. As expected, the HD cohort as a whole had a reduction in the basal ganglia BPnd to approximately 50% of that seen in HC. On follow-up scans, [(18)F]MNI-659 uptake declined in the putamen and caudate nucleus in all 8 participants. The mean annualized rates of decline in signal in the caudate, putamen, and globus pallidus and the putamen were 16.6%, 6.9%, and 5.8%, respectively. In HC, the annualized reduction in signal in striatal regions was less than 1%. CONCLUSION: Longitudinal data in this small cohort of participants with early HD support [(18)F]MNI-659 PET imaging of PDE10 as a useful biomarker to track HD disease progression.

Convenient synthesis of 18F-radiolabeled R-(-)-N-n-propyl-2-(3-fluoropropanoxy-11-hydroxynoraporphine.

Aporphines are attractive candidates for imaging D2 receptor function because, as agonists rather than antagonists, they are selective for the receptor in the high affinity state. In contrast, D2 antagonists do not distinguish between the high and low affinity states, and in vitro data suggests that this distinction may be important in studying diseases characterized by D2 dysregulation, such as schizophrenia and Parkinson's disease. Accordingly, MCL-536 (R-(-)-N-n-propyl-2-(3-[(18)F]fluoropropanoxy-11-hydroxynoraporphine) was selected for labeling with (18)F based on in vitro data obtained for the non-radioactive ((19)F) compound. Fluorine-18-labeled MCL-536 was synthesized in 70% radiochemical yield, >99% radiochemical purity, and specific activity of 167 GBq/µmol (4.5 Ci/µmol) using p-toluenesulfonyl (tosyl) both as a novel protecting group for the phenol and a leaving group for the radiofluorination.

The phosphodiesterase 10 positron emission tomography tracer, [18F]MNI-659, as a novel biomarker for early Huntington disease.

IMPORTANCE: In Huntington disease (HD) striatal neuron loss precedes and predicts motor signs or symptoms. Current imaging biomarkers lack adequate sensitivity for assessing the early stages of HD. Developing an imaging biomarker for HD spanning the time of onset of motor signs remains a major unmet research need. Intracellular proteins whose expression is altered by the mutant huntingtin protein may be superior markers for early HD stages. OBJECTIVE: To evaluate whether [18F]MNI-659 (2-(2-(3-(4-(2-[18F]fluoroethoxy)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione), a novel phosphodiesterase 10 positron emission tomography (PET) ligand, is a sensitive marker for striatal changes in early HD. DESIGN, SETTING, AND PARTICIPANTS: A cohort of individuals with HD, including premanifest (pre-HD) or manifest with motor signs (mHD), underwent clinical assessments, genetic determination, [18F]MNI-659 PET imaging, and brain magnetic resonance imaging. Age-matched healthy volunteers (HVs) also received clinical assessments and PET and magnetic resonance imaging. MAIN OUTCOMES AND MEASURES: Binding potentials (BPnds) were estimated for brain regions of interest, specifically within the basal ganglia, and compared between participants with HD and the HVs and correlated with markers of HD severity and atrophy of basal ganglia nuclei. RESULTS: Eleven participants with HD (8 mHD and 3 pre-HD) and 9 HVs participated. Ten of 11 HD participants had known huntingtin CAG repeat length, allowing determination of a burden of pathology (BOP) score. One individual with HD declined CAG determination. All participants with mHD had relatively early-stage disease (4 with stage 1 and 4 with stage 2) and a Unified Huntington's Disease Rating Scale (UHDRS) total Motor subscale score of less than 50. The HD cohort had significantly lower striatal [18F]MNI-659 uptake than did the HV cohort (mean, -48.4%; P < .001). The HD cohort as a whole had a reduction in the basal ganglia BPnd to approximately 50% of the level in the HVs (mean, -47.6%; P < .001). The 3 pre-HD participants had intermediate basal ganglia BPnds. Striatal [18F]MNI-659 uptake correlated strongly with the severity of disease measured by the clinical scale (UHDRS Motor subscale; R = 0.903; P < .001), the molecular marker (BOP; R = 0.908; P < .001), and regional atrophy (R = 0.667; P < .05). CONCLUSIONS AND RELEVANCE: As a promising striatal imaging biomarker, [18F]MNI-659 is potentially capable of assessing the extent of disease in early mHD. Furthermore, [18F]MNI-659 may identify early changes in medium spiny neurons and serve as a marker to predict conversion to mHD. Additional studies with larger, stratified cohorts of patients with HD and prospective studies of individuals with pre-HD are warranted.

Automated one-step radiosynthesis of the CB1 receptor imaging agent [(18) F]MK-9470.

The fluorine-18-labeled positron emission tomography (PET) radiotracer [(18) F]MK-9470 is a selective, high affinity inverse agonist that has been used to image the cannabinoid receptor type 1 in human brain in healthy and disease states. This report describes a simplified, one-step [(18) F]radiofluorination approach using a GE TRACERlab FXFN module for the routine production of this tracer. The one-step synthesis, by [(18) F]fluoride displacement of a primary tosylate precursor, gives a six-fold increase in yield over the previous two-step method employing O-alkylation of a phenol precursor with 1,2-[(18) F]fluorobromoethane. The average radiochemical yield of [(18) F]MK-9470 using the one-step method was 30.3 ± 11.7% (n = 12), with specific activity in excess of 6 Ci/µmol and radiochemical purity of 97.2 ± 1.5% (n = 12), in less than 60 min. This simplified, high yielding, automated process was validated for routine GMP production of [(18) F]MK-9470 for clinical studies.

In vivo evaluation of 18F-MNI698: an 18F-labeled radiotracer for imaging of serotonin 4 receptors in brain.

UNLABELLED: Serotonin 4 receptors (5-hydroxytryptamine receptor 4 [5HT4R]) hold promise as a novel therapeutic approach to multiple brain disorders, including Alzheimer and Huntington disease. In vivo imaging of these receptors with selective 5HT4R radiotracers and PET would be valuable to investigate alterations in 5HT4R in different brain disorders and to assist drug discovery. In this study, (18)F-MNI698 was evaluated as a potential PET radiotracer for imaging of 5HT4R in the brain. METHODS: Eighteen PET studies were performed in 3 adult rhesus monkeys. The radiotracer was administered as a bolus intravenous injection or bolus plus constant infusion (time that would be required to inject the bolus at the infusion rate = 60 min), and arterial blood was collected for data quantification. Kinetic models were used to estimate distribution volumes and binding potentials, for which the cerebellum was used as a reference region. (18)F-MNI698 test-retest variability and upper mass dose limits were determined. Preblocking studies using several doses of SB204070, a selective 5HT4R antagonist, were performed. RESULTS: (18)F-MNI698 avidly entered the monkey brain (peak percentage injected dose of ∼ 6.6%), and its brain distribution was consistent with known 5HT4R densities. At 120 min after bolus injection and after the start of radiotracer infusion, only less than 5% and approximately 10% parent compound was present in blood, respectively. Measured binding potentials were underestimated by 22%-36% when noninvasive methods were used for data quantification in comparison with invasive methods. A good agreement was found between test-retest measurements. The radiotracer upper mass dose limit (<5% occupancy) was determined to be 13.1 µg per 70 kg of body weight. SB204070 blocked the radiotracer binding in a dose-dependent manner. CONCLUSION: Data indicate that (18)F-MNI698 is a promising PET radiotracer for imaging of 5HT4R in the brain, and human studies are warranted based on these study results.

Whole-body biodistribution and dosimetry estimates of a novel radiotracer for imaging of serotonin 4 receptors in brain: [¹8F]MNI-698.

INTRODUCTION: A new radiotracer for imaging the serotonin 4 receptors (5-HT4) in brain, [¹8F]MNI-698, was recently developed by our group. Evaluation in nonhuman primates indicates the novel radiotracer holds promise as an imaging agent of 5-HT4 in brain. This paper aims to describe the whole-body biodistribution and dosimetry estimates of [¹8F]MNI-698. METHODS: Whole-body positron emission tomography (PET) images were acquired over 240 minutes after intravenous bolus injection of [¹8F]MNI-698 in adult rhesus monkeys. Different models were investigated for quantification of radiation absorbed and effective doses using OLINDA/EXM 1.0 software. RESULTS: The radiotracer main elimination route was found to be urinary and the critical organ was the urinary bladder. Modeling of the urinary bladder voiding interval had a considerable effect on the estimated effective dose. Normalization of rhesus monkeys' organs and whole-body masses to human equivalent reduced the calculated dosimetry values. The effective dose ranged between 0.017 and 0.027 mSv/MBq. CONCLUSION: The dosimetry estimates, obtained when normalizing organ and whole-body weights and applying the urinary bladder model, indicate that the radiation doses from [¹8F]MNI-698 comply with limits and guidelines recommended by key regulatory authorities that govern the translation of radiotracers to human clinical trials. The timing of urinary bladder emptying should be considered when designing future clinical protocols with [¹8F]MNI-698, in order to minimize the subject absorbed doses.

Synthesis and biological evaluation of positron emission tomography radiotracers targeting serotonin 4 receptors in brain: [18F]MNI-698 and [18F]MNI-699.

Two new benzodioxane derivatives were synthesized as candidates to image the serotonin 4 receptors by positron emission tomography (PET) and radiolabeled with fluorine-18 via a two-step procedure. Competition binding assays demonstrated that MNI-698 and MNI-699 had sub-nanomolar binding affinities against rat striatal 5-HT4 receptors (Ki of 0.20 and 0.07 nM, respectively). PET imaging in rhesus monkey showed that the regional brain distribution of [(18)F]MNI-698 and [(18)F]MNI-699 were consistent with the known densities of 5-HT4 in brain. [(18)F]MNI-698 and [(18)F]MNI-699 are among the first fluorine-18 radiotracers developed for imaging the 5-HT4 receptors in vivo and are currently under preclinical investigation in primates for future human use.

An automated module for the separation and purification of cyclotron-produced 99mTcO4-.

INTRODUCTION: The shortage of reactor-produced molybdenum-99 ((99)Mo, t(½)=66 h) has renewed interest in alternative production methods of its daughter isotope, technetium-99m ((99m)Tc, t(½)=6.02 h). While adsorption chromatography serves as a mechanism for selective elution of sodium pertechnetate from technetium generators, this method of purification is not sufficient for many alternative production methods. Several ion-separation/solid phase extraction chromatography methods are known, yet none have been demonstrated on cyclotron-produced [(99m)Tc]TcO(4)(-). Herein we describe the design, manufacture and optimization of a remotely operated module for the purification of sodium pertechnetate from a bulk solution of molybdate. METHODS: The automated purification module was designed to separate [(99m)Tc]TcO(4)(-) using either Dowex 1x8 or an Aqueous Biphasic Extraction Chromatography (ABEC) resin. (100)Mo composite targets were irradiated with 18.5 MeV protons for 10 µA·h using an ASCI TR19 cyclotron. Once purified, the radiopharmaceutical quality of (99m)TcO(4)(-) isolated from each process (Dowex and/or ABEC) was established by assaying for molybdate breakthrough, alumina levels and, in the case of the Dowex approach, residual organics. RESULTS: The separation processes are efficient (75% for Dowex, 90% for ABEC) and complete in less than 30 min. Overall, up to 2.1 GBq of (99m)Tc was produced using the (100)Mo(p,2n)(99m)Tc transformation, processed using the separation module and subjected to a detailed chemical and radionuclidic analysis. Due to its expense and limited availability, (100)MoO(4)(2-) was recovered in >90% yield using a precipitation/filtration/lyophilization approach. CONCLUSIONS: Na[(99m)Tc]TcO(4) was produced using a medical cyclotron, recovered using an automated purification module and found to exceed all established quality control parameters.

Chemoenzymatic synthesis and enzymatic analysis of 8-modified cytidine monophosphate-sialic acid and sialyl lactose derivatives.

The sialic acids found on eukaryotic glycans have remarkably diverse core structures, with a range of modifications at C5, C7, C8 and C9. These carbohydrates have been found to play key roles in cell-cell interactions within eukaryotes and often serve as the initial site of attachment for viruses and bacteria. Consequently simple changes to the structures of the sialic acids can result in profoundly different and often opposing biological effects. Of particular importance are modifications at the 8-position. These include O-acetylation, carried out by an acetyl transferase, and particularly polysialylation, catalyzed by a polysialyltransferase. As part of a structural and mechanistic study of sialyltransferases and polysialyltransferases, access was needed to sialic acid-containing oligosaccharides that are modified at the 8-position of the sialic acid to render this center non-nucleophilic. The free 8-modified sialic acid analogues were synthesized using a concise, divergent chemical synthetic approach, and each was converted to its cytidine monophosphate (CMP) sugar donor form using a bacterial CMP-sialic acid synthetase. The transfer of each of the modified donors to lactose by each of two sialyltransferases was investigated, and kinetic parameters were determined. These yielded insights into the roles of interactions occurring at that position during enzymatic sialyl transfer. A transferase from Campylobacter jejuni was identified as the most suitable for the enzymatic coupling and utilized to synthesize the 8''-modified sialyl lactose trisaccharides in multimilligram amounts.

A new sialidase mechanism: bacteriophage K1F endo-sialidase is an inverting glycosidase.

Bacteriophages specific for Escherichia coli K1 express a tailspike protein that degrades the polysialic acid coat of E. coli K1 that is essential for bacteriophage infection. This enzyme is specific for polysialic acid and is a member of a family of endo-sialidases. This family is unusual because all other previously reported sialidases outside of this family are exo- or trans-sialidases. The recently determined structure of an endo-sialidase derived from bacteriophage K1F (endoNF) revealed an active site that lacks a number of the residues that are conserved in other sialidases, implying a new, endo-sialidase-specific catalytic mechanism. Using synthetic trifluoromethylumbelliferyl oligosialoside substrates, kinetic parameters for hydrolysis at a single cleavage site were determined. Measurement of kcat/Km at a series of pH values revealed a dependence on a single protonated group of pKa 5. Mutation of a putative active site acidic residue, E581A, resulted in complete loss of sialidase activity. Direct 1H NMR analysis of the hydrolysis of trifluoromethylumbelliferyl sialotrioside revealed that endoNF is an inverting sialidase. All other wild type sialidases previously reported are retaining glycosidases, implying a new mechanism of sialidase action specific to this family of endo-sialidases.

Episulfonium ion-mediated cyclic peptide and triazine synthesis.

The product of an episulfonium ion-mediated cyclotrimerisation, previously reported as being a 15-membered ring trilactam, has now been shown to be a 1,3,5-triazine. Smaller medium-ring bilactams have, however, been synthesised from linear precursors using the sulfur-based methodology.

Selective five- and six-membered cyclic amine syntheses via capture of episulfonium ions.

Amide nitrogens open episulfonium ions to form pyrrolidines or piperidines selectively, depending on the nitrogen substituent, in either reversible or irreversible reactions.